The metabolism of the thyroid hormones by monolayer cultures of monkey hepatocarcinoma cells was studied further. Uptake of the hormones was shown to be by passive transport, but varied with the iodine content of the molecule. Propylthiouracil inhibited phenolic ring deiodination but not nonphenolic ring deiodination, thus accounting for increased accumulation of reverse T3. Investigation of cell homogenates showed that the two deiodinases had different pH optima, but were both stimulated by sulfhydryl compounds, and were present in the particulate fraction. This cell culture is a useful model system in which to study thyroid hormone metabolism.